Chloride channels comprise essential components of many important biological processes, including regulation of cell volume, secretion and reabsorption of epithelial fluid, stabilization of membrane resting potential in muscle, acid secretion by gastric parietal cells, and signal transduction. Moreover, an increasing number of pathologies, such as cystic fibrosis and a certain heritable myotonias, have been associated with chloride channel dysfunctions. However, structure-function relationships within this class of transmembrane macromolecules are only beginning to be elucidated. This lack of knowledge is due, at least in part, to our inability to identify high-affinity blockers of this channel class. Such blockers have been crucial to our understanding of structure and function in a variety of other channels, notably those involved in cation translocation in excitable tissues. The overall goal of this application is to characterize at the molecular level the interaction of chlorotoxin, a newly discovered, high-affinity blocker of gastric, voltage-regulated C-l channels, with its target site, and to use the information acquired to begin to develop a 3-dimensional model for the pore region of this protein. The principal investigator has developed an E. coil expression system for chlorotoxin, and demonstrated that the fully active polypeptide can be purified to homogeneity from bacterial extracts. We now propose to: (1) obtain a complete kinetic characterization of the effects of chlorotoxin on the rabbit gastric chloride channel; (2) characterize the binding of chlorotoxin to the gastric channel biochemically and electrophysiologically; (3) employ chlorotoxin as a photoaffinity ligand for identification of pore-forming sequences of the channel; (4) map chlorotoxin residues which contribute to channel binding, using a combined chemical modification-site directed mutagenesis strategy and (5) identify unique chloride channel residues which interact with specific sites on chlorotoxin (as identified above) using site-directed mutagenesis of the cloned gastric chloride channel. Successful execution of these aims will provide a well-characterized, high-affinity blocker of gastric, and perhaps additional, chloride channels, knowledge on the nature and distribution of its molecular targets, identification of pore-forming sequences and a preliminary and experimentally testable model for the structure of the pore itself.